Our main goal is to define the ORI (origin of DNA replication) sequences needed for DNA puffs II/9A and II/2B in the fungus fly Sciara coprophilia, and to understand the relationship between ecdysone induced initiation of amplification and transcriptional enhancers. DNA puff ORI will be mapped by chromosome walks across II/9A and II/2B coupled with quantitative Southern blots to map the region of maximum amplification for each puff. A newly developed 2-D gel method will be applied to each ORI domain for finer resolution mapping; this method will also show if the same ORI sites are used for prepuff replication (prior to amplification). The DNA sequence of ORI at both puffs will be determined and compared to one another Transcription units active during DNA puffing will be mapped by Northern blots with probes spanning the chromosome walks at both DNA puff loci, and their 5' start sites determined by primer extension. Transcriptional enhancers will be located by P element transformation in Drosophila using CAT assays. DNase I hypersensitivity and in vivo DMS footprinting of enhancer regions will locate binding sites for trans-acting proteins, and these areas will be sequenced. Do enhancers overlap ORI? Future studies will address whether the activated ecdysone receptor directly binds to upstream sites to initiate both amplification and transcription. Gene amplification is an important feature of some cancers, and DNA puffs provide an excellent model system to understand the mechanism of amplification and its hormonal control.